Restoration of differentiated functions in multicellular aggregates of a human liver epithelial cell line

Whether or not the differentiated functions of a SV40 T antigen-immortalized human liver epithelial cell line (THLE-5b), most of which were lost after serial subcultivations, are restored was examined by culturing it in a three-dimensional system using a macroporous membrane material, expanded polytetrafluoroethylene (ePTFE). The morphology and differentiated functions of THLE-5b cells were influenced by the modification of the ePTFE surface. THLE-5b cells formed anchored multicellular aggregates on a PVA-coated ePTFE surface 3 to 4 days in culture, but formed cell monolayers on a PVA-uncoated ePTFE surface as did the cells on a type I collagen-coated polystyrene surface. Albumin and transferrin were expressed in multicellular aggregates, but was negative in the corresponding monolayer cultures. However, α1-antitrypsin was not expressed in multicellular aggregates as well as monolayer cultures. Although cytokeratin 18 was found both in monolayer cultures and in multicellular aggregates, cytokeratin 19 and γ-glutamyltranspeptidase were negative in monolayers and in multicellular aggregates. The present data suggest that some of the differentiated functions of THLE-5b cells are restored by three-dimensional cell–cell interaction in multicellular aggregates and also suggest that the multicellular aggregates of THLE-5b, as immobilized on a PVA-coated ePTFE surface, will provide us a model system for the development of a bioreactor using hepatocytes, particularly for a hybrid-type artificial liver.